Phosphoinositides suppress γ-secretase in both the detergent-soluble and -insoluble states

Abstract

γ-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized γ-secretase assay system, we previously found that γ-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of γ-secretase. Recently, level of secreted β-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on γ-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized γ-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered γ-secretase activity. PI(4,5)P2 was also found to inhibit γ-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of β-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoin ositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4-8 mM. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, γ-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate γ-secretase activity by suppressing its association with the substrate. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.